Determination of genotoxic impurity in atazanavir sulphate drug substance by LC–MS

doi:10.1016/j.jpba.2016.09.025

Journal of Pharmaceutical and Biomedical Analysis

Volume 132, 5 January 2017, Pages 156-158

https://doi.org/10.1016/j.jpba.2016.09.025 Get rights and content

Highlights

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A sensitive LC–MS method for GTI-A, genotoxic impurity in atazanavir sulphate drug was developed.
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Method validation was done in respect of QL, DL, linearity and accuracy.
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Method: can be used in Quality control laboratories of pharmaceutical industry if and when required.

Abstract

A sensitive LC–MS method was developed for the determination of tert-butyl 2-[4-(pyridine-2-yl) benzyl] hydrazine carboxylate (GTI-A), a genotoxic impurity in Atazanavir sulphate drug substance. The method was validated as per International Council for Harmonization guidelines, for QL, DL, linearity and accuracy. The QL and DL values obtained were 1.1 ppm and 0.3 ppm respectively. The Correlation coefficient found for the linearity study was 0.999. The % recovery of the added impurity in the range of 96.4–100.4 ensured the accuracy of the method.

Introduction

The international regulatory bodies from various regions have been emphasized the determination of impurities in the drug substances and drug products because of their toxicological concern [1]. Genotoxic impurities are those have potential to cause cancer [2]. These impurities (GTIs) carry to a drug substance in the manufacturing process as starting materials, reagents, intermediates, by-products, degradants, etc. [3]. If impurity contains structural alert for mutagenicity, it can be considered as a genotoxic impurity [4]. The European Medicines Agency released guidelines on the control of GTI and ICH also released guidelines on Genotoxic impurities [ICH M7] [4], [5]. These guidelines proposed a threshold of toxicological concern value (1.5 μg/day). The limit for the determination of these impurities was set based on the maximum daily dose (MDD) of the drug. Atazanavir sulphate (Fig. 1a) is an HIV-1 protease inhibitor (PI) and sold under the trade name Reyataz. The chemical name of atazanavir is [methyl N-[(1S)-1-{[(2S,3S)-3-hydroxy-4-[(2S)-2-[(methoxycarbonyl)amino]-3,3-dimethyl-N'-{[4-(pyridin-2-yl) phenyl]methyl}butane hydrazido]-1-phenylbutan-2-yl]carbamoyl}-2,2-dimethylpropyl] carbamate]. Its molecular formula is C₃₈H₅₂N₆O_7•H₂SO₄, which corresponds to a molecular weight of 802.9 (sulfuric acid salt). Tert-butyl 2-[4-(pyridine-2-yl) benzyl] hydrazine carboxylate (GTI-A) (Fig. 1b) is a chemical used in atazanavir sulphate process at early stage. Since there is a structural alert, it is essential to control and prove that this material is not carried to final stage. Atazanavir sulphate is available in different dosages 150, 200 and 300 mg. Based on the daily dosage, the limit of GTI-A was fixed as 3.8 ppm. The authors attempted to develop an LC–MS method for the determination of GTI-A and validated.

Section snippets

Chemicals and reagents

The Atazanavir sulphate Drug substance was obtained from Hetero Drugs limited (R&D), Hyderabad, India. The GTI-A (Tert-butyl 2-[4-(pyridine-2-yl) benzyl] hydrazine carboxylate) was obtained from Rampex labs (P) ltd, Hyderabad, India. Ammonium formate (AR grade) formic acid (AR grade), high pure acetonitrile and methanol (HPLC grade) were obtained from Merck Life sciences, Mumbai, India.

Instrumentation

LC–MS analysis was carried out on LC–MS 2010 EV single quadrupole mass spectrometer having LCMS solutions

Method development

The limit for GTI-A in atazanavir sulphate was identified as 3.8 ppm based on the MDD of the drug. There are some literature reports available for the determination of process related and degradant impurities in atazanavir sulphate [6]. No literature is available for the determination of GTI-A, the authors are attempted to develop and validate the method for the determination of GTI-A in atazanvir sulphate. Initially the trials were carried out using HPLC UV method with phosphate buffer and

Conclusion

The findings of our current studies prove that the developed and validated method for the determination of tert-butyl 2-[4-(pyridine-2-yl)benzyl] hydrazine carboxylate (GTI-A), a genotoxic impurity in atazanavir sulphate drug substance is a sensitive LC–MS method in respect of QL, DL, linearity and accuracy. This method can be used in Quality control laboratories of pharmaceutical industry if and when required.

Acknowledgements

The authors are grateful to the management of Hetero Drugs Ltd., Hyderabad for their support. We would also like to express our appreciation to the authorities of Vignan University for constant encouragement.

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Short communicationDetermination of genotoxic impurity in atazanavir sulphate drug substance by LC–MS

Highlights

Abstract

Introduction

Section snippets

Chemicals and reagents

Instrumentation

Method development

Conclusion

Acknowledgements

Trends Anal. Chem

Reg. Toxicol. Pharmacol.

J. Pharm. Biomed. Anal.

Short communication
Determination of genotoxic impurity in atazanavir sulphate drug substance by LC–MS